Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal!

Grimsey, Natasha L.; Goodfellow, Catherine E.; Scotter, Emma L.; Dowie, Megan J.; Glass, Michelle; Graham, Scott E.

doi:10.1016/j.jneumeth.2008.02.014

Cited by 113 publications

(76 citation statements)

References 29 publications

(48 reference statements)

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“…This has also been pointed out by multiple investigators (e.g. Strater et al, 2001;Bodei et al, 2009;Jensen et al, 2009;Herber et al, 2004;Grimsey et al, 2008;Kirkpatrick, 2009;Herrera et al, 2013;Bohmer et al, 2014;Baker, 2015;Van Liefferinge et al, 2016).…”

Section: Introductionsupporting

confidence: 55%

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

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Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045–2064

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Section: Introductionsupporting

confidence: 55%

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

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“…Given the strong mRNA expression of CB 2 receptor in human eosinophils, it is possible that they were involved in the previously observed CB 2 expression by neutrophils (48)(49)(50). Additionally, it is noteworthy that the use of CB receptor Abs can be misleading (50,51). Our data show that freshly isolated human neutrophils do not significantly express CB receptors (mRNA) and are not activated by the CB receptor agonists used in the current study, and the effects of 2-AG are not prevented by CB receptor blockade.…”

Section: Discussionmentioning

confidence: 99%

The Endocannabinoid 2-Arachidonoyl-Glycerol Activates Human Neutrophils: Critical Role of Its Hydrolysis and De Novo Leukotriene B4 Biosynthesis

Chouinard

Lefebvre

Navarro

et al. 2011

The Journal of Immunology

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Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B4 biosynthesis, or LTB4 receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB4 biosynthesis, and an autocrine activation loop involving LTB4 receptor 1.

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“…The study of CB1-R relies on the availability of highly specific antibodies (Grimsey et al, 2008). Thus, CB1R…”

Section: Methodsmentioning

confidence: 99%

Cannabinoid Receptors in the Bed Nucleus of the Stria Terminalis Control Cortical Excitation of Midbrain Dopamine CellsIn Vivo

Massi¹,

Elezgarai²,

Puente³

et al. 2008

J. Neurosci.

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The endocannabinoid system is involved in multiple physiological functions including reward. Cannabinoids potently control the activity of midbrain dopamine cells, but the contribution of cortical projections in this phenomenon is unclear. We show that the bed nucleus of the stria terminalis (BNST) efficient relays cortical excitation to dopamine neurons of the ventral tegmental area (VTA). Anatomical and in vivo electrophysiological evidence demonstrate that excitatory projections arising exclusively from the infralimbic cortex converge on BNST neurons, which in turn project to and excite Ͼ80% VTA dopamine cells. At the ultrastructural level, cannabinoid type 1 receptors are detected within the BNST on axon terminals arising from the infralimbic cortex. We found that intra-BNST infusion of a cannabinoid agonist inhibits the firing of dopamine cells evoked by stimulation of the infralimbic cortex. Our data identify a new neuronal substrate for the actions of cannabinoids in the reward pathway.

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Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal!

Cited by 113 publications

References 29 publications

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

The Endocannabinoid 2-Arachidonoyl-Glycerol Activates Human Neutrophils: Critical Role of Its Hydrolysis and De Novo Leukotriene B4 Biosynthesis

Cannabinoid Receptors in the Bed Nucleus of the Stria Terminalis Control Cortical Excitation of Midbrain Dopamine CellsIn Vivo

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